Conidial Morphology Research Articles

Colletotrichum fioriniae infecting invasive Japanese hop (Humulus scandens) in the United States.

Japanese hop (Humulus scandens) is a non-native, invasive plant that colonizes disturbed riparian areas throughout the eastern United States and Canada, forming dense, monocultural stands that displace native plant communities due to a high reproductive rate, rapid growth, climbing bines, and dense shading (Balogh and Dancza 2008). It is capable of serving as a reservoir for agronomically important plant pathogens, such as the Tomato spotted wilt virus and powdery mildew species that infect commercial hemp and hop fields (Yoon et al. 2018; Weldon et al. 2020). In the spring of 2016, diseased populations of H. scandens were observed along the Monocacy River in Frederick County, Maryland with severe chlorotic and necrotic leaf lesions. Symptomatic leaves were surface sterilized and placed in moist chambers at 25°C for sporulation. Sporulating acervuli, lacking setae, developed on irregular, tan necrotic leaf lesions following 7 to 12 days in a moist chamber (Figure 1). Conidia were hyaline, aseptate, smooth-walled, fusiform to cylindrical with both ends acute (Figure 1B). Conidia measured (n = 100) [L x W; Average (+ Std. Err), range]: 12.42 µm (± 0.10), 8.41 - 14.48 µm; x 3.91 µm (±0.03), 3.03 - 4.91 µm. Monoconidial fungal cultures were obtained by transferring conidia with a sterile glass needle to acidified potato dextrose agar and incubated at 25°C for 2 to 3 days. Based on phenotypic characteristics and conidial morphology and size, the pathogen appeared to belong to the Colletotrichum acutatum complex (Damm et al. 2012). Therefore, six loci (ITS, GADPH, CHS1, HIS3, ACT, and TUB2) were amplified and sequenced from a representative isolate, 16-008, for species characterization (GenBank accessions MW023070 to MW023075) (Damm et al. 2012). For the ITS region and ACT, GADPH, and CHS1 loci, isolate 16-008 was 100% identical to C. fioriniae and shared 99% similarity to TUB2 and HIS3 for multiple accessions of C. fioriniae in GenBank. Gene sequences were aligned, trimmed, concatenated, and analyzed against 32 reference strains, within the C. acutatum complex (Damm et al. 2012). Concatenated loci were used to generate a maximum likelihood phylogeny using W-IQ-TREE (Trifinopoulos et al. 2016). Results from the phylogenetic analysis demonstrated that isolate 16-008 was most genetically similar to C. fioriniae with a bootstrap support of 100% (Figure 2). Based on phenotypic and sequence analyses, isolate 16-008 was identified as C. fioriniae. Humulus scandens seedlings from Maryland (n = 3) were inoculated with a conidia suspension (107 conidia mL-1) with 0.125% Tween 20® and applied with an atomizer until runoff. Inoculated plants were placed in a dew chamber at 25°C for 2 days. Experimental plants were distributed in a mist tent at 25°C with 14 h of light and monitored for 2 weeks. Negative control plants (n = 2) were sprayed with a sterile 0.125% Tween 20® water solution. All inoculated plants were symptomatic by 12 days post inoculation. No symptoms were observed on the mock-inoculated plants. Symptoms were identical to disease field samples. Inoculations were repeated with the same results. Colletotrichum fioriniae was reisolated and confirmed from excised leaf lesions via ITS and ACT sequencing. To our knowledge, this is the first report of C. fioriniae naturally infecting H. scandens within the United States (Farr and Rossman 2020). Future studies will evaluate the host range of this isolate due to the species broad host range and the weed's extensive distribution.

First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky.

In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar 'White CBG' being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-5.8 µm (n=50). The strains produced blue-black fertile perithecia on carrot agar when induced according to the method of (Bowden and Leslie, 1999). To confirm pathogenicity, flowers of hemp cultivars 'Lifter', 'Trump Towers', 'Wife' and 'White CBG' were inoculated in the greenhouse with a representative fungal strain (20Hemp010). Plants were inoculated at two different stages: when the styles were still green or after they had become senescent. Macroconidia were collected from 7- to 9-day-old cultures grown under a 12-hour light-dark cycle. Plants were spray-inoculated with a 5 x 105 per ml conidial suspension in 0.05% Tween 20 until runoff. Flower heads were individually covered with clear plastic bags and incubated for 72 h at 95-100% humidity under greenhouse benches to avoid direct light. Bags were removed after 72 h and returned to the bench. Greenhouse conditions were 23-25°C with a 14-hour photoperiod and 50% RH. Symptoms developed 7 dai in 1% of the flowers inoculated when styles were green, and 36% of the flowers that had senescent styles. Symptoms were similar to those initially noticed in Boyle County, including necrotic flower bracts and sugar leaves, and visible fungal growth. Symptoms were more severe on plants inoculated when styles were necrotic. Recovered fungi were morphologically similar to 20Hemp010. Genomic DNA was extracted from the mycelium with the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit. A fragment of the translation elongation factor 1-alpha 1 gene was amplified with primers EF1 and EF2 as described by (O'Donnell et al. 1998). Amplicons were sequenced and the consensus (MZ407909) was compared with the NCBI GenBank Refseq database by BLASTn. The top hit was Fusarium graminearum with 100% identity (JF270185.1). Pairwise alignments via MycoBank Fusarium MLST and Fusarium-ID also revealed a top hit of F. graminearum with 100% identity (AY452957.1). Conidial and colony morphology were also consistent with F. graminearum (Leslie and Summerell, 2006), thus we conclude that this species was the causal agent of the flower blight and mold. The same disease was subsequently confirmed on hemp in Breathitt and Franklin Counties in KY in 2020. This is the first report of this disease in KY, although F. graminearum has been reported previously causing a similar flower blight on hemp in NY and NC (Bergstrom et al., 2019, Thiessen et al. 2020). Fusarium graminearum is common in KY as a cause of Fusarium head blight on wheat and Gibberella ear rot on corn. In cereals, fungal infection is facilitated by the production of the mycotoxin deoxynivalenol (DON), which is harmful to humans and livestock (Desjardins and Hohn, 1997). As hemp production in Kentucky continues to rise for production of CBD products and edible grains, accumulation and concentration of DON in these products could become a concern.

Alternaria leaf spot of broccoli caused by Alternaria alternata in Bangladesh

This study aimed to isolate and characterise the pathogen associated with Alternaria leaf spot on broccoli and to evaluate the inhibitory effects of fungicides against it. We isolated and identified the fungal pathogen as Alternaria sp. using morphological and cultural methods. Based on the aligned sequences of the internal transcribed spacer (ITS) and molecular phylogenetic analysis by the neighbour-joining method, the isolates (Ab1 and Ab2) were confirmed as Alternaria alternata. The conidia of the isolates were dark brown, cylindrical, obclavate to muriform. The conidiophores were olivaceous brown, septate, and branched. The conidial morphology of the isolates ranged from 52.4–92.4 × 10–20 μm with 2–6 transverse and 0–3 longitudinal septa. Both isolates yielded positive results in the pathogenicity test on broccoli leaves by developing brown and circular spots with concentric rings on the leaves surrounded by yellow halos. The culture studies revealed that the maximum growth of the pathogen was obtained at 30 °C and pH 6.0. Tilt 250 WC showed the highest potential in suppressing the mycelial growth of the A. alternata in vitro at a concentration as low as 50 µg/mL. The results from this study contributed to the positive identification of the pathogen and characterised A. alternata as a destructive pathogen of broccoli which may be successfully controlled by the fungicide Tilt.

First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan.

First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan.

First Report of Leaf Spot Caused by Colletotrichum siamense on Philodendron bipinnatifidum in Guangxi, China.

Philodendron bipinnatifidum belongs to the Araceae family, as its graceful plant architecture and the function of purifying air and water, which has important ornamental value (Yu et al. 2019). P. bipinnatifidum is often cultivated in parks and along roadsides. In August 2019 and June 2020, leaf spot disease was observed on over 80% of P. bipinnatifidum plants in Qingxiushan Park (N22°47'23.35″, E108°23'4.26″), Nanning, Guangxi, China. The disease was also frequently observed on P. bipinnatifidum in some other places in Nanning. Symptoms began as small, round, brown spots. As the disease developed, the center of the lesions was sunken with a dark brown border (Fig. 1). Under severe conditions, some spots were joined into larger irregular spots, and even whole leaves died. For fungal isolation, small pieces (5 × 5 mm) were cut from the margin between lesion and healthy tissues of symptomatic leaves, disinfected with 75% ethanol for 10 s, 1% sodium hypochlorite solution for 1 min and washed by sterile water for three times. Over one hundred morphologically similar colonies with white mycelia and a dark green pigment were obtained after 5 days incubation on potato dextrose agar (PDA) at 25°C. Isolates GBZ6-1, GBZ9-1 and GBZ9-2 were selected for intensive study, and the mycelial growth rates of them averaged 14.5 mm/day, 14.6 mm/day and 13.2 mm/day, respectively. Isolates could produce orange conidia on PDA for 7 days. Conidia were elliptical, aseptate and colourless, with sizes of 15.4 ± 0.14 µm × 5.8 ± 0.1 µm, 15.6 ± 0.14 µm × 5.3 ± 0.1 µm and 15.0 ± 0.16 µm × 6.3 ± 0.1 µm for GBZ6-1, GBZ9-1 and GBZ9-2, respectively. Appressoria were mostly brown ovoid, conidial appressoria averaged 7.62-9.31 μm × 5.84-742 μm, and mycelial appressoria were 8.06-9.22 μm × 6.28-6.92 μm. Genomic DNA was extracted by the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the rDNA internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012) were sequenced. Phylogenetic analysis was performed using RAXML (Version 2.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, and GAPDH). The results showed that three isolates were identified as C. siamense (accessions MW116805-MW116816, for ACT, CAL, CHS-1 and GAPDH of GBZ6-1, GBZ9-1 and GBZ9-2; MW073391-MW073393 for ITS of GBZ6-1, GBZ9-1 and GBZ9-2). According to the cultural and conidial morphology, as well as phylogenetic analysis, these isolates were identified as Colletotrichum siamense. Pathogenicity tests were conducted on one-year-old plants. Young healthy leaves of three plants were artificially wounded by gently scratching with a sterile needle and 10 µl of conidial suspension (106 spores/ml) were applied to per wound site for each isolate. Wounded leaves were inoculated with 10 µl of water as controls. All plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 15 days at 28 °C, whereas no symptoms were observed on the control leaves. To fulfill Koch's postulates, fungi were reisolated from symptomatic leaves and morphologically identical to the inoculated isolates (Xue et al. 2020). To our knowledge, this is the first report of leaf spot caused by C. siamense on P. bipinnatifidum worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. siamense on P. bipinnatifidum.

First Report of Pestalotiopsis microspora Causing Leaf Blight of Banana in Bangladesh.

Banana (Musa sp.) is one of the most important fruit crops in the world. It is also a very popular fruit crop and cultivated widely in Bangladesh. Banana diseases are one of the limiting factors for its production. Recently, a new banana leaf blight was detected in various districts of Gazipur, Bangladesh, with an incidence of 5 to 10% in June 2020 and 15 to 20% in January 2021. The initial symptoms of the disease were narrow dark brown lesions (2 to 5 x 5 to 12 mm) that later became irregular brown spots (10 to 15 x 5 to 7 mm). The lesions gradually spread from the middle of the leaf to the margin and dark brown spots appeared on the diseased leaves. There was a clear golden yellowish boundary around the spot. Blighted tissue often covered one-thirds to one-half of the infected leaves. To isolate the pathogen, 100 diseased leaves were collected from five different locations in Gazipur, Bangladesh. Small pieces (5x5 mm) were cut from the margins of lesions and surface sterilized with 75% ethanol for 30 s followed by 1% NaClO for 1 min. The samples were then rinsed three times with sterile distilled water, dried on sterilized filter paper, and placed on 1% water agar at 25°C for 7 days. Hyphal tips were then excised, placed on PDA and incubated at 25°C for 7 days. From the 20 isolates obtained, the colony characteristics and conidial morphology of three isolates, BLS_BU1, BLS_BU2, and BLS_BU3, were examined. The colony of all of three isolates was white, cottony, and circular in growth. The acervuli were black and covered all the culture plate. Conidia of all the isolates were 5-celled, fusiform, and wider in the middle than the apical and basal cells. The three internal cells were olivaceous, concolourous, and slightly constricted at the septae; the terminal cells were hyaline (Shi et al. 2015). The conidium measured 20.3 ± 1.2 to 22.7 ± 1.8 by 7.2 ± 0.6 to 8.6 ± 0.4 μm. The mean lengths of the two to four hyaline apical appendages were 20.5 to 30.25 μm and the hyaline basal appendage was 6.1 to 7.6 μm (Han et al. 2019). DNA was extracted from the fungal colony using a DNeasy Plant Mini Kit (Qiagen). PCR was performed with primers ITS1/ITS4 ITS region and Sanger sequenced by Macrogen (Seoul, Korea) (White et al. 1990). Isolates BLS_BU1 (MH707065), BLS_BU2 (MH707065), and BLS_BU3 (MH712283) had a sequence similarity of over 99% with GenBank reference Pestalotiopsis microspora (MH707065) of National Center for Biotechnology Information (NCBI) database. The reconstructed phylogenetic tree also confirmed their phylogenetic position. To assess pathogenicity, the leaves of four 2-month-old healthy plants were needle wounded and inoculated with a spore suspension (106 spores/ ml) harvested from 10-day-old cultures of isolate BLS_BU1. As a control treatment, five leaves of two healthy plants were inoculated with sterile distilled water. Treated plants were maintained in the field with average temperature ranging from 28°C to 30°C and relative humidity from 75% to 80%. After 2 weeks, all inoculated leaves produced characteristic symptoms similar to those observed in the field. However, the control plants did not develop any symptoms. Pestalotiopsis microspora from the inoculated leaves was successfully reisolated, thus confirmed that P. microspora was the cause of leaf blight of banana. To our knowledge, banana appears to be a new host of P. microspora. this This is the first report of leaf blight of banana caused by P. microspora in Bangladesh.

First Report of Alfalfa Anthracnose Caused by Colletotrichum americae-borealis in Xinjiang, China.

Alfalfa (Medicago sativaL.) is widely planted in the world as one of the most important leguminous forage crops, and it is also the first choice of forage crops for animal feed in Xinjiang. In June 2018, alfalfa plants with typical anthracnose symptoms were observed in 75% of alfalfa fields in Hutubi County, Xinjiang, China. The disease usually occurred in alfalfa fields that had been planted for more than 2 years and was distributed in patches in the field. The incidence rate ranged from 7.5% to 53%, and the fatality rate ranged from 0 to 3%. Greater incidence was observed in fields with older stands. At the early stage of disease, pale brown prismatic or oval sunken lesions with dark brown to black edges were observed at the base of the stem of alfalfa plants. As the symptoms progressed, lesions on stems turned necrotic, and the center of the lesion became gray-white with black dots. In severe cases, the lesion expanded around the stem, causing the upper part of the stem to break off, or wilt and die. Twenty plant stem sections with typical symptoms were sampled and surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 1 min, rinsed in sterilized distilled water, dried on sterilized filter paper for 45 s, placed on potato dextrose agar (PDA), and incubated in the dark at 25°C for 7 days. A fungus was frequently isolated from the surface-sterilized segments, and the colonies of this fungus were white and flat at first, and later the center of colonies became pale brown with black microsclerotia (2.0~3.2 mm. n = 30) and white or brown acervuli (1.0~1.8 mm. n = 30). A large number of conidia and setae spread from ruptured microsclerotia under microscopy. Conidia (n = 40) were hyaline, smooth-walled, straight, aseptate, cylindrical to fusoid, both tips acute to round, 13.7 to 19.5×3.0 to 4.5 μm . Setae (n = 30) were dark brown to black, smooth-walled, 3~6 septate, straight or slightly curved, 66.9~185.1 μm long, tip round and base swollen, 3.9~5.2 μm width. Sometimes setae formed directly on hyphae or brown acervuli. Colony and conidia morphology were similar to the description of Colletotrichum americae-borealis (Damm et al. 2014; Lyu et al. 2020). DNA was extracted from fresh mycelia of three representative isolates (R11, R12 and R13) and the ITS, ACT, CHS-1 and HIS3 genes of three isolates were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences of three isolates were identical, and twelve aligned sequences from three representative isolates were deposited in GenBank (MT877442, MT877443 and MT877444 for ITS, MW854350, MW854351 and MW854352 for ACT, MW270930, MW270931 and MW270107 for CHS-1, MW854347, MW854348 and MW854349 for HIS3). Sequence analysis revealed that the ITS, ACT, CHS-1 and HIS3 sequences of three representative isolates were shared 99% (355/356 bp for HIS3) to 100% (550/550 bp for ITS, 261/261 bp for ACT, 221/221 bp for CHS-1) identities to each sequence of an American strain (CBS 136232) of C. americae-borealis from alfalfa in GenBank (NR160760 for ITS, KM105434 for ACT, KM105294 for CHS-1, KM105364 for HIS3). Four phylogenetic trees were constructed by the Mrbayes method (Damm et al. 2014), and the result showed that three representative isolates grouped with C. americae-borealis. Combined with morphological observation and molecular biological identification, the pathogen was identified as C. americae-borealis. Pathogenicity tests were executed twice on alfalfa seedlings in a greenhouse. Pots containing ten 40-day old seedlings (Xinjiang daye) were sprayed with a 100 ml of condial suspension (10^6 condia/ml) of R11. Control pots were sprayed with 100 ml of sterile distilled water. Two weeks after inoculation under greenhouse conditions (25 ± 2°C, 12-h photoperiod, 85% humidity), brown spots and necrotic lesions developed on the stem of inoculated alfalfa seedlings, which were similar to disease plants in fields, and C. americae-borealis was reisolated from symptomatic tissue. The control seedlings remained symptomless. Anthracnose caused by C. americae-borealis was reported on alfalfa in the north region of America and Iran (Damm et al. 2014; Alizadeh et al. 2015), as well as Gansu, Inner Mongolia, Yunnan and Heilongjiang Province of China ( Xu. 2019; zhang et al. 2020) . To our knowledge, this is the first report of Colletotrichum americae-borealis causing Alfalfa Anthracnose in Xinjiang, China. This finding can provide an important reference for understanding the distribution and control of this disease.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

Cultural, Morphological Characterization and Aggressiveness of Alternaria alternata Causing Leaf Spot of Soybean

Background: Alternaria leaf spot caused by Alternaria alternata is one of the most important and destructive disease of soybean causing severe yield loss in all soybean growing areas of southern and eastern part of Rajasthan. Successful management of Alternaria leaf spot is mainly dependent on accurate and efficient detection of pathogen, amount of genetic and pathogenic variability present in pathogen population. The main reason for frequent “breakdown” of effective resistance is the variability that exists in the pathogen population, which necessitates a continual replacement of cultivars due to disease susceptibility. Methods: The twelve fungal isolates randomly were collected from six districts of major soybean growing part of Rajasthan i.e., Udaipur, Chittorgarh, Pratapgarh, Kota, Baran and Jhalawar. The culture was purified single spore techniques. These were then further compared among each other for any variations in cultural characters, colour of colonies, Growth rate, conidial morphology and pathogenic variability. Result: Twelve different isolates of A. alternata were obtained in pure culture and characterized for cultural, morphological variation and aggressiveness of this fungus varied in their cultural characters, colour of colonies, growth rate of isolates, conidial morphology and isolates also exhibited variations in incubation period, latent period, number and size of lesions were produced.

First report of the powdery mildew pathogen of hops, Podosphaeria macularis, naturally infecting cannabis (Cannabis sativa L., marijuana) plants under field conditions

Plants of the cannabis (Cannabis sativa L., marijuana) strain ‘Chronic Ryder’, grown outdoors in the Fraser Valley of British Columbia (BC), displayed symptoms of powdery mildew in late July 2019. The disease progressed rapidly under cool, wet conditions to infect leaves, stems, shoot tips and inflorescences by early September. To identify the pathogen, DNA was extracted from healthy and diseased leaves and subjected to PCR using eukaryotic universal primers that amplified the ITS1-5.8S-ITS2 region of rDNA. A 650 bp band present only in the diseased plants, showed 99.45% sequence homology to Podosphaeria macularis, the powdery mildew pathogen that infects hop plants. In greenhouse-grown cannabis infected with the powdery mildew pathogen Golovinomyces ambrosiae, a 700 bp size band was present. Conidial morphology also distinguished the two pathogens – P. macularis produced ovoid-oval conidia with fibrosin bodies, while G. ambrosiae produced cylindrical-oblong conidia with no fibrosin bodies. A survey of powdery mildew-infected hop fields and indoor cannabis growing facilities in different geographical locations in BC, showed that plants were infected exclusively by either P. macularis or G. ambrosiae, respectively. Inoculations using P. macularis-infected leaf segments placed on cannabis ‘Pink Kush’ leaves under high humidity at 22–26°C gave rise to small mildew colonies after 24–35 days, confirmed to be P. macularis by PCR. Development of P. macularis in cannabis plants was slower compared with G. ambrosiae. To date, only one outdoor location for cannabis has been confirmed to have P. macularis; the potential for spread to other sites or to indoor cultivation facilities is unknown.

The antioxidant methyl gallate inhibits fungal growth and deoxynivalenol production in Fusarium graminearum

Production of the Fusarium toxin deoxynivalenol (DON) is associated with oxidative stress and has been indicated to be part of an adaptive response to oxidative stress in the important wheat fungus Fusarium graminearum. In this study, we found that the antioxidant methyl gallate (MG) displays inhibitory effects against mycelial growth, conidial formation and germination, and DON biosynthesis in F. graminearum in a dose-dependent manner. Treatment with 0.05% (w/v) MG resulted in an abnormal swollen conidial morphology. The expression of the TRI genes involved in DON biosynthesis was significantly reduced, and the induction of Tri1-GFP green fluorescence signals in the spherical and crescent-shaped toxisomes was abolished in the MG-treated mycelium. RNA-Seq analysis of MG-treated F. graminearum showed that 0.5% (w/v) MG inhibited DON production by possibly altering membrane functions and oxidoreductase activities. Coupled with the observations that MG treatment decreases catalase, POD and SOD activity in F. graminearum. The results of this study indicated that MG displays antifungal activity against DON production by modulating its oxidative response. Taken together, the current study revealed the potential of MG in inhibiting mycotoxins in F. graminearum.Graphical abstract

Diversity of Colletotrichum Species Associated with Olive Anthracnose Worldwide.

Olive anthracnose caused by Colletotrichum species causes dramatic losses of fruit yield and oil quality worldwide. A total of 185 Colletotrichum isolates obtained from olives and other hosts showing anthracnose symptoms in Spain and other olive-growing countries over the world were characterized. Colony and conidial morphology, benomyl-sensitive, and casein-hydrolysis activity were recorded. Multilocus alignments of ITS, TUB2, ACT, CHS-1, HIS3, and/or GAPDH were conducted for their molecular identification. The pathogenicity of the most representative Colletotrichum species was tested to olive fruits and to other hosts, such as almonds, apples, oleander, sweet oranges, and strawberries. In general, the phenotypic characters recorded were not useful to identify all species, although they allowed the separation of some species or species complexes. ITS and TUB2 were enough to infer Colletotrichum species within C. acutatum and C. boninense complexes, whereas ITS, TUB2, ACT, CHS-1, HIS-3, and GADPH regions were necessary to discriminate within the C. gloesporioides complex. Twelve Colletotrichum species belonging to C. acutatum, C. boninense, and C. gloeosporioides complexes were identified, with C. godetiae being dominant in Spain, Italy, Greece, and Tunisia, C. nymphaeae in Portugal, and C. fioriniae in California. The highest diversity with eight Colletotrichum spp. was found in Australia. Significant differences in virulence to olives were observed between isolates depending on the Colletotrichum species and host origin. When other hosts were inoculated, most of the Colletotrichum isolates tested were pathogenic in all the hosts evaluated, except for C. siamense to apple and sweet orange fruits, and C. godetiae to oleander leaves.

Digitodesmium polybrachiatum sp. nov., a new species of Dictyosporiaceae from Brazil

Digitodesmium is a genus of saprobic fungi, generally associated with decaying wood in freshwater habitats or in the soil. Fungi in this genus produce cheiroid, euseptate conidia on sporodochia. During an exam of a necrotic robusta coffee stem sent from Nova Venécia, state of Espírito Santo, to the Plant Clinic at the Universidade Federal de Viçosa (Brazil), for disease diagnosis, a fungus, recognized as having the typical features of Digitodesmium, was observed. The fungus was isolated in pure culture and DNA was extracted. Sequences of the partial 18S ribosomal RNA gene, large subunit of the nrDNA, internal transcribed spacer, and translation elongation factor 1-α were generated. The combination of results of the phylogenetic analysis with the exam of the morphology led to the conclusion that the fungus from the dead coffee stems represents a monophyletic distinct lineage within Digitodesmium and an undescribed species for the genus. The concatenate tree also revealed that Digitodesmium is divided in two distinct clades. The novel species can be differentiated morphologically from other species of Digitodesmium by the size of the conidia, the number of arms, and the presence of appendages. The new species Digitodesmium polybrachiatum is hence proposed herein. A comparative table of conidial morphology for the species in the genus is also included.

Outbreaks of crown rot in Fragaria x ananassa caused by Neopestalotiopsis mesopotamica in Ecuador

Strawberry crown rot is one of the most important diseases in Ecuador, with an incidence of up to 70% in the field. In the world, the strawberry crown rot is caused by different etiological agents, which makes efficient control of the disease difficult. In this study, 12 samples of strawberry plants were collected, at different stages of disease development, to determine the causative agent in Ecuador. The fungal isolates, consistent with the isolation, were identified and characterized using molecular and morphological approaches, and finally, Koch's postulate was carried out. Based on the form of growth, mycelial color, conidial morphology, and molecular characteristics (sequencing of the ITS and EF-1α region), the pathogen associated with the symptoms showed 99% similarity with the species Neopestalotiopsis mesopotamica. The result of Koch's postulates showed symptoms similar to those observed in field plants, in addition, the same inoculated isolate was recovered from artificially inoculated tissues. This is the first report of this fungus causing strawberry crown rot in Ecuador and in the world.

Response of balsam poplar to inoculation with Marssonina balsamiferae from Minnesota, USA

AbstractLeaf spots of poplar (Populus) species and hybrids in North America commonly are caused by three Drepanopeziza species (previously referred to by their asexual morph names in genus Marssonina). A fourth species, Marssonina balsamiferae, is known only from herbarium specimens and was described from balsam poplar (Populus balsamifera) leaves collected in 1966 in Manitoba, Canada. Balsam poplar leaves collected in 2014 in Minnesota, USA bore spots with acervuli and conidia consistent with those of M. balsamiferae, and isolates were obtained from these leaves. Conidial morphology and analysis of nuclear ribosomal ITS sequences distinguished the Minnesota strains from the very common leaf spot pathogen of poplars in the northcentral region of the USA, Drepanopeziza brunnea. Inoculation of rooted balsam poplar cuttings with conidia of M. balsamiferae strains from Minnesota resulted in necrotic leaf spots. Acervuli bearing conidia consistent with those of M. balsamiferae were observed on symptomatic leaves, and the fungus was reisolated from infected tissues. Contemporary recognition of existence and pathogenicity of M. balsamiferae, including presence in the USA, should prompt further investigation of its geographic distribution and relationships with hosts and related fungal species.

Transcription factor UvMsn2 is important for vegetative growth, conidiogenesis, stress response, mitochondrial morphology and pathogenicity in the rice false smut fungus Ustilaginoidea virens

Transcription factors (TFs) play critical roles in the control of development and pathogenicity of phytopathogens by directly or indirectly regulating the expression of downstream genes. Here, we identified and characterized a zinc finger TF UvMsn2 in Ustilaginoidea virens, a homolog of MoMsn2 from the rice blast fungus. Heterogenous complementation assay revealed that UvMsn2 fully restored the defects of the ∆Momsn2 mutant in vegetative growth, conidiation and pathogenicity. Deletion of UvMsn2 in U. virens led to a reduction of the pathogen in vegetative growth, aerial hyphae and conidiation. Additionally, the ∆Uvmsn2 mutant displayed defects in conidial morphology and germination, as well as mitochondrial morphology. Pathogenicity and toxicity assays revealed that the ∆Uvmsn2 mutant was non-pathogenic and less inhibitory to rice seed germination. The ∆Uvmsn2 mutant showed different sensitivity to various stresses. Further microscopic examination found that UvMsn2 was localized in both cytosol and nucleus, and translocated from cytosol to nucleus under the treatment of NaCl. Our results demonstrate that UvMsn2 is a critical TF that regulates the vegetative growth, conidiogenesis, stress response, mitochondrial morphology and virulence in the rice false smut fungus.

Isolation and Characterization of Nickel-Tolerant Trichoderma Strains from Marine and Terrestrial Environments.

Nickel contamination is a serious environmental issue that requires immediate action. In this study, 23 strains of Trichoderma were isolated from terrestrial and marine environments and identified using a polyphasic approach of morphological characterization and ITS gene sequence analysis. The Trichoderma strains were tested for their tolerance and biosorption of nickel. Our results showed the growth of all Trichoderma strains on Trichoderma Selective Medium (TSM) with 50–1200-ppm nickel, indicating their tolerance of this heavy metal even at a relatively high concentration. Six Trichoderma strains (three isolated from terrestrial substrates and three from marine substates) had the highest radial growth on TSM with 50-ppm Ni. Among these fungal isolates, Trichoderma asperellum (S03) isolated from soil exhibited the best growth after 2 days of incubation. For the biosorption of nickel, the accumulation or uptake efficiency by the six selected Trichoderma was determined in Potato Dextrose Broth (PDB) supplemented with 50-ppm Ni using a Flame Atomic Absorption Spectrophotometer (AAS). The percent uptake efficiency of the three strains of T. asperellum (S03, S08, and LL14) was computed to be up to 66%, while Trichoderma virens (SG18 and SF22) and Trichoderma inhamatum (MW25) achieved up to 68% uptake efficiency. Observation of the Trichoderma strains with Scanning Electron Microscopy (SEM) before and after the absorption of nickel showed very minimal damage on the hyphal and conidial surface morphology, but changes in the colonial characteristics were observed. Our study highlighted the potential of terrestrial and marine strains of Trichoderma for the bioremediation of nickel pollution.

Identification and characterization of Colletotrichum species associated with anthracnose disease of banana

AbstractBanana (Musa spp.) is one of the five most abundantly produced fruits in the world and is widely planted in tropical and subtropical areas. Banana anthracnose is one of the main diseases during the growth and postharvest storage period of banana, seriously affecting quality and production. In this study, 24 samples of banana anthracnose were collected near the cities Nanning, Qinzhou, Baise, and Chongzuo in Guangxi Province, China. Based on colony features, conidial and appressorial morphology, and sequence analysis of several genomic regions (internal transcribed spacer [ITS] region, glyceraldehyde‐3‐phosphate dehydrogenase [GAPDH], actin [ACT], β‐tubulin [TUB2], chitin synthase [CHS‐1], calmodulin [CAL], and the intergenic region of apn2 and MAT1‐2‐1 [ApMAT]), the 32 Colletotrichum isolates obtained were identified as five species: C. fructicola (41%), C. cliviicola (28%), C. siamense (16%), C. karstii (9%), and C. musae (6%). A conidial suspension (106 spores/ml) was used to inoculate banana seedlings for pathogenicity tests by applying 20 μl to wound sites. Lesions caused by C. musae developed most rapidly while those of C. karstii took the longest time to develop. This is the first report of C. siamense, and C. karstii associated with banana anthracnose in China, and the first report of C. fructicola and C. cliviicola associated with banana anthracnose worldwide.

Fusarium solani and Lasiodiplodia pseudotheobromae, fungal pathogens causing stem rot disease on durian trees (Durio zibethinus) in Eastern Thailand

Durian (Durio zibethinus) is known as the king of fruit in Southeast Asia (Bampenrat et al., 2020), and is one of the most economically important export fruits in Thailand (Charoensumran et al., 2021). However, a major problem affecting durian plantations in Thailand is stem rot disease. Symptoms of the infected trees are bark and trunk canker, twig blight, yellow leaves, and wilting. Thirty symptomatic bark and wood samples were collected in November 2020 from different durian orchards in Rayong and Chanthaburi Provinces in the Eastern Thailand (Fig. 1). The samples were cut into 1×1 cm fragments, surface sterilised with 0.5% sodium hypochlorite solution, and washed three times in sterilised water similar to the method used by Chantarasiri et al. (2021). The sterilised samples were plated on dichloran Rose Bengal chloramphenicol (DRBC) agar as a selective medium and potato dextrose agar (PDA) as a relatively rich medium. The agar plates were incubated at 30°C for seven days under a 12-hour light/dark photoperiod. The isolated fungal colonies and hyphal tips were cut and placed on the PDA for culture purification. All fungal isolates were primarily categorised according to their colony and conidial morphology. The first fungal group had a white and cottony colony, producing slightly curved macroconidia with two-four septa (Fig. 2). The second group also had a white and woolly colony but these colonies grew rapidly, turned dark grey after seven days, and did not produce conidia on PDA (Fig. 3). DNA of representative fungal isolates was extracted using the GF Fungus DNA Extraction Kit (Vivantis, Malaysia) and PCR amplified with ITS1/ITS4 primers (White et al., 1990). The resulting PCR products were sequenced by Macrogen Inc. (South Korea) and deposited in the GenBank with Accession Nos. MZ461923, MZ461933, MZ493230, and MZ493231, respectively. Sequence alignment using BLASTn showed that the first fungal group was related to the Fusarium solani strain GP (MN658459.1) with 100% coverage and 95.81-98.99% identity. The second group was related to the Lasiodiplodia pseudotheobromae isolate KEGA_11 (MK922068.1) with 99–100% coverage and 96.64-98.94% identity. Phylogenetic analysis using SeaView version 5.0.2 software confirmed the sequence alignment results (Figure 4). One hundred milliliters of each fungus cultured in potato dextrose broth was inoculated to the trunk of durian seedlings and placed in a glasshouse at ambient temperature for seven days. The same volume of sterilised water was inoculated for the controls. The experiment was performed in triplicate. White mycelia and brown lesions were visually observed in the inoculated seedlings, whereas the controls remained healthy. The fungi were re-isolated and identified by PCR as F. solani and L. pseudotheobromae, thus fulfilling Koch's postulates. This report provides evidenced that F. solani and L. pseudotheobromae are the fungal pathogens causing stem rot disease on durian trees. Previous reports described these fungi causing stem rot in hackberry and mango (Khanzada et al., 2004; Liang et al., 2020). To date, Phytophthora sp. has been attributed as the cause of stem rot disease on durian trees in Thailand. Misidentification of the stem rot fungal pathogens could result in the use of inappropriate methods to control the disease. The authors would like to thank the orchardists in Rayong and Chanthaburi Provinces for kindly providing the samples used in this study.

Cellular uptake of chitosan and its role in antifungal action against Penicillium expansum

Chitosan has wide-spectrum antimicrobial activity but knowledge of its antifungal mechanism is still incomplete. In this study, transcriptome of Penicillium expansum upon chitosan treatment was analyzed by RNA-Seq. KEGG enrichment analysis revealed that endocytosis as well as other physiological pathways was regulated by chitosan treatment. Clathrin adaptor protein mu-subunit (PeCAM) gene, which encodes a protein associated with clathrin-dependent endocytosis, was up-regulated after chitosan treatment. Deletion of PeCAM resulted in changes of conidial, hyphal and colonial morphology. Confocal microscopy images of the distribution of fluorescein isothiocyanate-labeled chitosan confirmed cellular internalization of chitosan. However, deletion of PeCAM almost completely blocked uptake of chitosan into fungal cells and ΔPeCAM mutant exhibited less sensitivity to chitosan compared with wild type, suggesting that chitosan uptake is mediated by clathrin-dependent endocytosis and internalized chitosan also plays an important role in its antifungal activity. Collectively, our results provide a new insight into the antifungal mechanism of chitosan.